Cytochrome c oxidase (COX) comprises 13 subunits: 3 encoded in mitochondrial DNA (mtDNA), ten in the nucleus. Of the ten nuclear subunits, three (subunits VIa, VIIa, and VIII) have muscle-specific isoforms. The mitochondrial genes are known to evolve ten times faster than single copy nuclear genes in higher primates. Thus, COX provides an ideal system in which to determine how the increased mutation rate in mtDNA affects the mutation rate of the nuclear genes. The long-term goal of this project is tousle evolutionary (sequence) comparisons to assess the function of COX nuclear-coded subunits and the regulation of COX nuclear genes. By determining which regions of selected COX genes change only very slowly and thus appear evolutionarily constrained, and which regions have evolved rapidly, consistent with acquiring new or altered functions. The specific aims in pursuit of this goal are: Aim 1. To determine when in the evolution of higher primates the replacement substitutions observed in the human. COX4 gene occurred, and whether or not these changes are due to interaction with COX subunit II. Aim 2. To determine when in primate evolution the changes in the COX5B, COX7X and COX7AL gene occurred. Aim 3. To use the method of phylogenetic footprinting to identify conserved cis-regulatory sequence elements in the regulatory regions of both constitutively expressed and tissue- specific CO:X nuclear genes. Aim 4. To investigate the role of gene duplication on the evolution of the mammalian COX complex by determining the time of the gene duplication leading to the tissue-specific genes COX6AH COX7AH, and COX8H. Aim 5. To determine the rates of evolution of intron 1 of the COX6AH gene from coding sequence in the yeast homologue. Because COX is the terminal enzyme complex of the mitochondrial electron transport chain, it is critically important for oxidative metabolism in aerobic tissues. COX deficiency has been identified as the molecular defect in several types of mitochondrial myopathy and encephalomyopathy. Applying evolutionary approaches to define function and regulation of mammalian COX nuclear genes will provide insights into subunit function and tissue-specific gene regulation, particularly of the muscle-specific isoforms. These insights are critical to our understanding of this essential enzyme complex and will enable us eventually to design rational therapy for patients with these molecular defects.